This invention generally relates to the sterilization of materials, especially polymeric materials for use in biomedical applications, such as polymeric particles for drug delivery or polymeric implants.
The use of polymeric materials in biomedical applications is a rapidly developing technology, especially the use of polymeric particles in controlled drug delivery. Sterilization of these particles is essential to their safe use in vivo. The most common methods for sterilization employ ethylene oxide, UV-radiation, .gamma.-irradiation, or superheated steam. Each of these methods possess certain disadvantages if applied to sterilization of materials for biomedical applications. For example, ethylene oxide residual gas may remain in the material, which can cause hemolysis (Clark, et al., J. Surg. 36 (1966); Nair, J. Biomat. Appl., 10:121 (1995)) or other toxic reactions. Other solvents, whether employed as a sterilant or merely as a process medium, similarly may leave residues that must be removed prior to use in a human. Gamma radiation may damage polymers, for example, by altering their shear and tensile strength, or elasticity. The use of UV- or .gamma.-radiation or high temperatures (as, for example, during autoclaving) also may reduce the quality of encapsulated drug or other labile biological materials, especially labile protein drugs, by decreasing bioavailability or by modifying chemical groups to decrease bioactivity.
Hydrostatic pressure (1200-8000 bar) has been used to mechanically rupture yeast cells and spores, as shown, for example, in Hayakawa, et al., J. Food Sci., 59:159 (1994); Hayakawa, et al., J. Food Sci., 59:164 (1994); and Hashizume, et al., Biosci. Biotech. Biochem., 59:1455 (1995). However, hydrostatic pressure coupled with very high shearing forces and high temperatures has met with limited success in sterilizing samples highly contaminated with spores. Konig, et al., "Autosterilization of Biodegradable Implants by Injection Molding Process", J. Biomed. Mater. Res., 38:115-19 (1997). The use of hydrostatic pressures at these magnitudes requires specially designed equipment capable of withstanding such pressures. This equipment generally is more costly to purchase and maintain than lower pressure operating systems. Extremely high pressures may also irreversibly deform some polymeric materials, which would be undesirable for many biomedical applications of these material.
High pressure or supercritical fluids, such as carbon dioxide, have been studied for use in sterilization of food. For example, Haas, et al., "Inactivation of Microorganisms by Carbon Dioxide Under Pressure," J. Food Safety, 2:253-65 (1989) describes numerous experiments using high pressure carbon dioxide (up to 900 psig (6 MPa)) to treat food products and several types of microorganisms. However, only up to four orders of magnitude reduction in living cells was provided with most of the microorganisms tested, i.e., complete sterilization was not achieved. Similarly, Kamihira, et al., "Sterilization of Microorganisms with Supercritical Carbon Dioxide," J. Biol. Chem. 51(2):407-12 (1987) describes efforts to sterilize microorganisms using supercritical carbon dioxide. However, sterilization of bacillus was not achieved. Nor was sterilization of a microorganism concentration of at least 10.sup.6 CFU/ml (colony forming units per milliliter) achieved as generally required in standard sterilization tests. See, e.g., Konig, et al., "Autosterilization of Biodegradable Implants by Injection Molding Process", J. Biomed. Mater. Res., 38:115-119 (1997); Dempsey, et al., "Sterilization of Medical Devices: A Review", J. Biomat. Appl. 3:454-523 (1989). Konig teaches that "the target range of contamination of 1.times.10.sup.5 to 5.times.10.sup.6 spores/g granules is comparable to the number of spores present on commercially available Paper Strip Biological Indicators used to qualify, validate, and monitor steam sterilization cycles" (p. 118). Similarly, Dempsey teaches using a "standardized challenge of 10.sup.6 bacillus subtilis spores" to evaluate sterilization efficiency (p. 463).
Supercritical fluid sterilization also has been explored in nonfood applications. U.S. Pat. No. 5,043,280 to Fischer, et al. describes the use of supercritical gases to sterilize polymeric carriers. However, the method disclosed therein uses organic solvents, which could be the actual sterilant and will leave solvent residues in the polymeric materials. Fischer also disclosed treating polymers for long periods of time (e.g., 12 hours) and at high temperatures (e.g., 50.degree. C.), conditions that may damage encapsulated biological materials. More importantly, Fischer began with a microorganism concentration of only 10.sup.4 CFU/ml, which is below the 10.sup.6 CFU/ml used in standard sterilization tests.
Some researchers claim to have achieved "sterilization" of several bacterial microbes using carbon dioxide based on only 2 to 3 log order microbial reduction, for example, starting with 10.sup.8 CFU/ml of bacterial cells and obtaining 10.sup.6 or 10.sup.5 CFU/ml viable cells following treatment, which would be reported as 99% or 99.9% inactivation. In contrast, standard sterilization tests generally (as described above) require that at least 10.sup.6 colony forming units per milliliter (CFU/ml) of starting material be used and that sterilization requires complete inactivation of these cells.
It is therefore an object of the present invention to provide a method and an apparatus to sterilize polymeric materials for use in biomedical applications, in the substantial absence of organic solvents and at relatively low temperatures for short durations.
It is a further object of the present invention to provide a method and an apparatus to completely inactivate a variety of microorganisms using supercritical fluid carbon dioxide.